Staining protocol for Imaging Mass Cytometry

Bake the slides at 60°C. Ensure that all visible wax has been removed

Dewax the slides in xylenes in the fume hood for 0h 10m 0s

Repeat step 2 and dewax the slides in fresh xylene in the fume hood for 0h 10m 0s

Hydrate the slides in descending grades of ethanol (100%, 95%, 80%, 70%) for 0h 5m 0s each.

Wash the slides in double distilled water for 0h 5m 0s in a Coplin jar placed on an orbital shaker plate with gentile agitation.

Perform antigen retrieval bu immersing slides in 1X epitope retrieval bugger (pH 9.0) for 0h 20m 0s in a steamer

Prepare 1X epitope retrieval buffer from its 10X stock solution.

Cool slides for 0h 40m 0s

Wash the slides in double distilled water for 0h 5m 0s in a Coplin jar with gentle agitation on an orbital shaker. Perform this step twice.

Wash the slides with 1X TBS for 0h 10m 0s with gentle agitation on an orbital shaker.

Use a PAP pen to draw an outline encircling the sample.

Block with 3% BSA in 1X TBS for 1h 0m 0s at room temperature in a hydration chamber.

You can use an empty pipette tip box where the slides rest on the tip shelf and the bottom is filled halfway with water.

Blocking solution should be diluted from 10% BSA freshly made from powder. The remaining 10% BSA should be aliquoted and stored at -20°C and diluted at time of use.

Use enough blocking solution to cover the section.

Prepare the antibody cocktail calculating the total volume of antibodies at the concentrations specific for the assay and bring the volume up to a final volume using 0.5% BSA. in 1X TBS.

Dilutions can be found on Table 1.

Place the slides in a hydration chamber and pipette the antibody mix on to the section.

Store the antibody cocktail mix on ice and add it on to the samples within 1-2 hours of preparation for best results.

Incubate overnight with the antibody cocktail at 4°C in a hydration chamber.

Wash the slides in 0.1% Triton X-100 in 1X TBS for 0h 5m 0s in Coplin jars with slow agitation on an orbital shaker.

Repeat step 15 two more times.

Wash the slides in 1X TBS for 0h 5m 0s with gentle agitation on an orbital shaker.

Repeat step 17.

Stain the tissue with Intercalator-Ir in 1X PBS (1:1000) for 1h 0m 0s at room temperature in a hydration chamber.

Wash the slides in double distilled water for 0h 5m 0s with gentle agitation on an orbital shaker.

Air dry the slides for at least 0h 20m 0s at room temperature.

Load the stained slide into the pre-tuned and pre-calibrated Hyperion IMC System.

Create a new project on the CyTOF software.

Draw a panorama that covers the tissue that will be ablated.

Determine regions of interest (ROIs) to ablate with increments less than 3mm² on the tissue, ensuring that cortical and medullary regions are separate within the panorama created. Regions with artificial section damage are excluded.

Create or select an antibody/metal template.

Make sure to select "generate txt file".

Save the project file and start ablation of tissue.

Once ablation is complete, load the file on MCD viewer to check the quality of each image and channel.